Enzyme substrate comprising a functional dye and associated technology and methods

Abstract

Enzyme substrates and associated technology of the present invention are provided. An enzyme substrate of the invention may comprise a biologically functional fluorescent dye and an enzyme-specific substrate moiety attached in such a way that the functionality of the functional dye is diminished. An enzymatic reaction may cleave at least a portion of the substrate moiety from the enzyme substrate to provide a more functional product dye. This product dye may be nonfluorescent or weakly fluorescent, in general, and relatively fluorescent, in a particular condition, such as when bound to a partner biological molecule or an assembly of partner biological molecules. An enzyme substrate of the present invention may thus be useful in fluorescence detection, and/or in any of a variety of useful applications, such as the detection of enzymatic activity in a cell-free system or in a living cell, the screening of drugs, or the diagnosis of disease.

The invention claimed is: 
     
       1. A substrate selected from a compound having a structural formula, DYE-(B) m , and a pro-enzyme substrate thereof, wherein DYE, when independent of DYE-(B) m , comprises a dye selected from a biologically functional fluorescent dye and a fluorogenic dye, the dye being capable of binding to a partner biomolecule, partner biomolecules, or an assembly of partner biomolecules; wherein m is selected from 1, 2, 3, 4 and 5; and wherein each of at least one B, independently, comprises an enzyme substrate moiety that is capable of enzymatic transformation comprising cleavage of a bond involving the dye and at least one B, cleavage of a bond within at least one B, or formation of a bond involving at least one B; and
 wherein the dye is a phenanthrodium dye having a structural formula, 
 
       
         
           
           
               
               
           
         
       
       wherein:
 B comprises a peptide or an amino acid; 
 at least one ligand of ligands R 1  and R 2  comprises B, where the C-terminal carbonyl of B is capable of forming an enzymatically cleavable peptide bond with an amino group of the dye; 
 if any one ligand of ligands R 1  and R 2  does not comprise B, said any one ligand comprises H; 
 ligand R 3  comprises a substituted or an unsubstituted C1 to C10 alkyl; 
 ψ comprises a cation selected from H+, Na+, K+, NH 4 +, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and 
 d is a number of ψ sufficient to render overall charge of the substrate neutral. 
 
     
     
       2. The substrate of  claim 1 , wherein m is selected from 1 and 2. 
     
     
       3. The substrate of  claim 1 , wherein at least one B is a peptide. 
     
     
       4. The substrate of  claim 1 , wherein the enzymatic transformation comprises phosphorylation catalyzed by a kinase. 
     
     
       5. The substrate of  claim 1 , wherein B comprises an enzyme substrate moiety for a peptidase enzyme. 
     
     
       6. The substrate of  claim 5 , wherein B comprises an enzyme substrate moiety for an enzyme selected from a caspase, a trypsin, a chymotrypsin, an elastase, a cathepsin B, and a cathepsin L. 
     
     
       7. The substrate  claim 1 , wherein when B comprises a charged peptide, ligand R 3  comprises a substituted or an unsubstituted C4 to a C10 alkyl. 
     
     
       8. The substrate of  claim 1 , wherein B comprises an enzyme substrate moiety for a caspase-3 enzyme and ligand R 2  comprises H. 
     
     
       9. The substrate of  claim 8 , wherein B comprises Z-DEVD (SEQ ID NO: 1).